SARS-CoV-2 Omicron infection augments the magnitude and durability of systemic and mucosal immunity in triple-dose CoronaVac recipients

ABSTRACT The inactivated whole-virion vaccine, CoronaVac, is one of the most widely used coronavirus disease 2019 (COVID-19) vaccines worldwide. There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron infection. In this prospective cohort study, 41 recipients of triple-dose CoronaVac and 14 unvaccinated individuals were recruited. We comprehensively profiled adaptive immune parameters in both groups, including spike-specific immunoglobulin (Ig) G and IgA titers, neutralizing activity, B cells, circulating follicular helper T (cTfh) cells, CD4+ and CD8+ T cells, and their memory subpopulations at 12 months after the third booster dose and at 4 and 20 weeks after Omicron BA.5 infection. Twelve months after the third CoronaVac vaccination, spike-specific antibodies and cellular responses were detectable in most vaccinated individuals. BA.5 infection significantly augmented the magnitude, cross-reactivity, and durability of serum neutralization activities, Fc-mediated phagocytosis, nasal spike-specific IgA responses, memory B cells, activated cTfh cells, memory CD4+ T cells, and memory CD8+ T cells for both the ancestral strain and Omicron subvariants, compared to unvaccinated individuals. Notably, the increase in BA.5-specific immunity after breakthrough infection was consistently comparable to or higher than that of the ancestral strain, suggesting no evidence of immune imprinting. Immune landscape analyses showed that vaccinated individuals have better synchronization of multiple immune components than unvaccinated individuals upon heterologous infection. Our data provide detailed insight into the protective role of the inactivated COVID-19 vaccine in shaping humoral and cellular immunity to Omicron infection. IMPORTANCE There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron breakthrough infection. In this prospective cohort study, the anti-severe acute respiratory syndrome coronavirus 2 adaptive responses were analyzed before and after the Omicron BA.5 infection. Our data provide detailed insight into the protective role of the inactivated COVID-19 vaccine in shaping humoral and cellular immune responses to heterologous Omicron infection. CLINICAL TRIAL This study is registered with ClinicalTrials.gov as NCT05680896.

campaign demonstrated outstanding effects in reducing the severity of SARS-CoV-2 infection (1,2).CoronaVac, a whole-virion inactivated vaccine produced by Sinovac, is the most widely offered COVID-19 vaccine globally.Although the diminished immunity and emergence of new viral variants have fueled an increase in the frequency of breakthrough infections, a real-world efficacy analysis in China showed that the protection efficacy of the triple-dose inactivated vaccine against BA.2 infection was 74% against pneumonia and 93% against severe COVID-19 (3).
Understanding the underlying immunological mechanism accounting for this vaccine-derived protection is not only crucial for next-generation COVID-19 vaccine optimization but also has important implications for future pandemic preparedness.In a prospective vaccine cohort, we previously analyzed the longitudinal immune responses elicited by the triple-dose inactivated COVID-19 vaccine for up to 2 months, includ ing serum-neutralizing antibody activity and B and T cell immune memory responses (4)(5)(6)(7).Since the adjustment of the public health policy for containing COVID-19 in China in December 2022, the subsequent SARS-CoV-2 Omicron variant wave resulted in large-scale breakthrough infection.In this study, we aimed to address two equally important issues.First, we examined whether CoronaVac-induced immune memory could still be effectively recalled up to 12 months after the third booster dose.The Omicron variant is associated with a marked capability to evade humoral immunity resulting from either prior natural infection or vaccination (8,9).There are rising concerns regarding the immune imprinting induced by ancestral strain-based vaccinations, which might compromise the antibody response to Omicron-based boosters.Therefore, we also attempted to investigate whether the immune responses elicited by triple-dose CoronaVac had any beneficial role in the anti-viral responses to subsequent Omicron infection.
In this study, we investigated these key questions by measuring SARS-CoV-2-spe cific dynamic immune trajectories, including antibody responses, Fc-mediated effector function, neutralization capacity, and cellular responses after Omicron BA.5 infection among recipients of triple-dose CoronaVac and unvaccinated individuals.Our data provide detailed insight into the favorable role of pre-existing immune responses elicited by triple-dose CoronaVac, which are capable of potently recalling SARS-CoV-2 immunity with cross-recognition to Omicron variants.

Study cohort and study design
We recruited a total of 55 participants, including 41 recipients of triple-dose CoronaVac and 14 individuals who were not vaccinated against COVID-19 (Fig. 1a).Omicron BA.5 infection was confirmed in all individuals by next-generation sequencing.The demo graphic and clinical characteristics of the cohort are summarized in Fig. 1b.The median age in the vaccinated group is 42.0 [95% confidence interval (CI) 30.0, 56.0], and the unvaccinated group is 45.0 (95% CI 31.5, 58.5).Out of 14 unvaccinated individuals, 3 (21.4%),but none of the vaccinated individuals, developed severe COVID-19 disease.Sampling at 12 months after the third-booster dose (T0), as well as at 2 weeks (T1) and 20 weeks (T2) after SARS-CoV-2 infection, allowed the immune trajectories to be measured to assess the establishment and maintenance of hybrid immunity.Simultaneous serum, peripheral blood mononuclear cell, and nasal swab samples were collected from each of the participants.The samples were subjected to a detailed analysis for both serological and cellular immune responses to SARS-CoV-2 antigens.The serum samples were tested for specific antibody titers using the enzyme-linked immunosorbent assay (ELISA) and neutralization activities against the ancestral, Delta, and Omicron BA.5 variants.We analyzed Fc-mediated phagocytosis activity to evaluate any functional differences in the antibody responses among vaccinated and unvaccinated individuals after BA.5 infection.

Breakthrough infection enhanced the magnitude and durability of humoral antibody responses
Antibodies are considered as the correlates of protection against SARS-CoV-2 infection (10).Therefore, we investigated the magnitude of variant-specific immunoglobulin (Ig)G and IgA binding in the serum.First, the binding of IgG and IgA with ancestral, Delta, BA.5, XBB.1, and XBB.1.5 was assessed by analyzing the serum collected before infection and at 2 and 20 weeks after SARS-CoV-2 infection (Fig. 2a and b).
The proportion of spike-specific memory B cells among the total memory B cells was also analyzed (Fig. 3c).Triple-dose CoronaVac recipients showed a median of 0.076%, 0.048%, 0.055%, and 0.041% memory B cells specific to ancestral, BA.1, BA.2, and BA.5, respectively, even at 12 months after the third booster vaccination.Memory B cells were absent in unvaccinated individuals at T0. BA.5 breakthrough infection expanded crossreactive spike-specific memory B cells.At T1, compared to unvaccinated controls, vaccinated individuals showed remarkably higher levels of memory B cells specific to the ancestral (0.170% vs 0.260%, 1.53-fold lower, P = 0.030), BA.5 (0.250% vs 0.430%, 1.72fold lower, P = 0.0009), BA.1 (0.170% vs 0.380%, 2.24-fold lower, P = 0.0003), and BA.2 (0.180% vs 0.200%, P = 0.451) strains (Fig. 3c and d).At T2, vaccinated individuals still maintained higher levels of spike-specific memory B cells than unvaccinated individuals, but the difference was not statistically different except for BA.5-specific memory B cells.Collectively, our data indicate that triple-dose CoronaVac established spike-specific memory B cells that could rapidly expand and proliferate into antibody-producing B cells upon breakthrough infection.
At T0, despite being 12 months after the third dose of CoronaVac, vaccinated individuals not only showed a detectable level of ancestral spike-specific memory cTfh cell responses (0.007%, 95% CI 0.003%-0.011%)but they also showed a slightly lower frequency of memory cTfh cells that were cross-reactive to the BA.1, BA.2, and BA.5 spikes, ranging from 0.004% to 0.005% (Fig. 3g).There was a trend toward slightly higher spike-specific memory cTfh cells in vaccinated individuals than in unvaccinated individuals, but no significant difference was observed (Fig. 3h).Collectively, our data suggest that breakthrough infection could quickly recall and activate specific cTfh cells, but memory cTfh cells were not significantly elevated compared with unvaccinated individuals.

BA.5 infection enhanced cross-reactive memory T cells among the recipients of triple-dose CoronaVac
T cells contribute to the defense against viral infections by orchestrating antibody production and cytotoxic killing of infected cells (18).Previously, we observed that CoronaVac induced durable, cross-reactive T cell responses (4,5).Given their importance, we examined whether BA.5 breakthrough infection could quickly recall and expand ancestral-and BA.5-specific CD4 + and CD8 + T cell responses.The data show that triple-dose CoronaVac recipients still showed detectable CD4 + T cells with a median frequency of 0.026% (95% CI 0.016%-0.039%)(Fig. 4a) and CD8 + T cells with a median frequency of 0.035% (95% CI 0.019%-0.056%)(Fig. 4b).In contrast, few spike-specific CD4 + or CD8 + T cell responses were detected in unvaccinated individuals.At T1, BA.5 breakthrough infection effectively expanded both CD4 + T cell (3.73-fold,P < 0.0001) and CD8 + T cell (5.45-fold,P < 0.0001) responses specific to the BA.5 spike (Fig. 4a and e).Similar trends were also observed in the T cell responses across the ancestral strain and the Omicron subvariants at T2.
The memory T cell subpopulation is critical to determine anti-viral responses upon antigen exposure.Therefore, we longitudinally measured the frequency of specific memory T cell responses.We found that compared with the corresponding baseline samples, BA.5 breakthrough infection led to augmented BA.5 spike-specific memory CD4 + T cell (5.24-fold,P < 0.0001) and memory CD8 + T cell (18.52-fold,P < 0.0001) responses at T1 (Fig. 4c and g).Interestingly, there was a trend toward a slightly higher frequency of specific memory CD4 + T cell responses in vaccinated individuals than in unvaccinated controls, but without significant differences (Fig. 4d).In contrast, compared with unvaccinated controls, vaccinated individuals showed substantially higher levels of ancestral (0.504% vs 0.139%, P = 0.001), BA.5 (0.500% vs 0.210%, P = 0.011), BA.1 (0.462% vs 0.091%, P = 0.002), and BA.2 (0.422% vs 0.216%, P = 0.040) spike-specific CD8 + memory T cell responses (Fig. 4g and h).To summarize, CD4 + and CD8 + memory T cells were still detectable 12 months after the third dose of CoronaVac.These pre-existing cross-reactive T cell responses and memory subsets could be effectively boosted and maintained by BA.5 infection.
The increase in BA.5-specific immune responses after breakthrough infection was consistently higher than for the ancestral strain Immune imprinting induced by ancestral strain-based vaccination might compromise the antibody response to Omicron-based boosters (8,9).Therefore, we compared the increases in the ancestral and BA.5-specific immune responses in vaccinated individuals (Fig. 5).Notably, we found that the increase from T0 to T1 in serum neutralization activities specific to BA.5 was comparable to that for the ancestral strain, while the enhancement from T0 to T1 in the BA.5 spike-specific IgG response was substantially higher than for the ancestral spike (Fig. 5a).Similarly, the majority of BA.5-specific immune parameters, including B cells, memory B cells, memory cTfh cells (Fig. 5b), CD4 + T cells, memory CD4 + T cells, and memory CD8 + T cells (Fig. 5c), displayed substantially greater fold changes than those specific to the ancestral strain at T1.

Distinct immune trajectories in breakthrough-infected individuals vs naturally infected individuals
The BA.5-infected vaccinated group exhibited remarkable divergence in SARS-CoV-2specific immune responses compared with the unvaccinated group.Therefore, further exploratory analyses were performed to gain a more detailed understanding of their fundamental differences.Following centering and scaling, hierarchical clustering of the participants revealed that vaccinated samples clustered away from the unvaccinated samples (Fig. 6a), highlighting the potent multicomponent immune memory induced by CoronaVac, even up to 12 months after the third CoronaVac dose.A multivariate partial least squares-discriminant analysis was performed across the vaccinated group and the unvaccinated group at the peak immune response after breakthrough infec tion (Fig. 6b).We found that vaccinated individuals could be perfectly separated from unvaccinated individuals.Notably, the immune profiles of participants in the vaccinated group exhibited selective enrichment of ancestral-specific ADCP, XBB.1-specific ADNP, BA.5-specific cTfh cells, and BA.1-specific B cells and memory B cells (Fig. 6c).
We also performed correlation analyses for the measured immune parameters, including antibody binding, neutralization activity, B cells, CD4 + T cells, CD8 + T cells, and cTfh cell responses, as well as their memory subsets, among the vaccinated group and the unvaccinated group.In the vaccinated group, we observed a moderate-to-low degree of correlation among the parameters defining the antibody response and among the parameters defining T cell responses.Furthermore, binding antibody responses were positively correlated with the CD4 + T cell response and the corresponding memory subsets (Fig. 6d).In the unvaccinated group, we observed a strong or moderate degree of correlation within the antibody compartment and within the T cell compartment.Nevertheless, a considerable proportion of inverse correlations were identified between antibodies and CD8 + memory T cells and cTfh cells, as well as between the CD4 + T cell response and memory cTfh cells (Fig. 6e).These results suggest that breakthrough infection had a well-integrated adaptive response between the antibody compartment and the T cell compartment, whereas the unvaccinated group harbored less coordinated immune trajectories between the antibody and T cell compartments, which might contribute to suboptimal antibody responses.
Additionally, we investigated the correlation between systemic and mucosal humoral immunity.Interestingly, BA.5-infected individuals in the vaccinated group demonstrated a significant positive correlation between serum-neutralizing activity and nasal IgA binding as well as IgG responses.Furthermore, strong positive correlations were identified within nasal IgA and IgG binding specific to SARS-CoV-2 variant spike antigens (Fig. 6f).Surprisingly, for previously unvaccinated individuals, serum-neutralizing activity was negatively correlated with nasal-binding antibody titers after BA.5 infection.Additionally, few cross-correlations were identified among the parameters of the nasal IgG-and IgA-binding antibodies (Fig. 6g).Collectively, our data highlight that vaccinated individuals have better synchronization of multiple immune components than unvacci nated individuals upon heterologous SARS-CoV-2 infection.

DISCUSSION
The inactivated whole-virion vaccine CoronaVac is one of the most widely used COVID-19 vaccines worldwide (19).Although there has been continual evolution of viral variants, which have managed to evade antibody responses to varying degrees, tripledose CoronaVac has retained more effectiveness against severe disease than against overall infection (20).Emerging evidence suggests that both T cells and antibody responses provide the greatest protection against infection and death in severe COVID-19 cases (21).In this study, we provide evidence of immune memory responses to ancestral and Omicron subvariants after BA.5 breakthrough infection among triple-dose CoronaVac recipients, which are maintained 12 months after the last dose.We also performed head-to-head comparisons of immune responses among vaccinated individuals vs unvaccinated individuals.When considered alongside the risk of severe infection and the long-term consequences of infection, our findings have important implications for vaccine policy.
Understanding the durability of the protection conferred by SARS-CoV-2 vaccination is of utmost importance to guide COVID-19 mitigation policies worldwide.The first key question we addressed is the durability of the immune memory and whether it is likely to protect against severe COVID-19 in the long term.We showed that serum anti-spike IgG and IgA responses were still detectable among the majority of triple-dose CoronaVac recipients at 12 months after the last dose.Additionally, we found that B and T cell responses after triple-dose CoronaVac were durable up to 12 months after the third dose, and that ancestral and Omicron subvariants were equally well recognized.Taken together, our longitudinal follow up over 12 months after the third CoronaVac dose demonstrated the durability and long-term maintenance of SARS-CoV-2 circulating B cells and CD4 + and CD8 + memory T cells.
The second key question that we attempted to address is the impact of immune imprinting from triple-dose CoronaVac on the adaptive response against Omicron subvariants.Our study analyzed breakthrough infection-induced humoral responses and cellular responses simultaneously, which allowed us to profile the dynamic kinetics of the antibody and cellular compartments.As the majority of people are vaccinated against the ancestral strain, immune imprinting induced by ancestral strain-based vaccination presents a major challenge to the performance of updated boosters.It has been suggested that boosting with a variant that is antigenically distinct from the ancestral strain would recall pre-existing memory B cells induced by the ancestral strain, preventing the generation of humoral immune responses targeting new variants (9,22).Intriguingly, our results did not show evidence of antigenic sin in the antibody responses after Omicron infection.We speculate this discrepancy might be largely due to the relatively longer interval (12 months) between breakthrough infection and the third CoronaVac (9,23,24).The long interval before Omicron breakthrough infection might preferentially recall cross-reactive antibodies that exhibit increased affinity and neutralization potency against Omicron, which could then drive a greater response given a subsequent antigen exposure (25).In addition, our finding aligns with recent evidence showing that either Omicron vaccination boosting or breakthrough infection not only triggers rapid memory cellular responses but also induces de novo B (26) and T cell (27) responses.Furthermore, we also compared the immune imprinting on T cell responses, which are less affected by viral variants than antibodies.This is likely due to the broader range of epitopes available to T cells compared with antibodies, where protective responses are more focused.Consistent with our findings, clinical and epidemiological studies also support our notion that any prior exposure to SARS-CoV-2 by infection and/or vaccination has a positive impact in mitigating the disease severity of subsequent infections (28).serum neutralization activity in vaccinated individuals (f) vs unvaccinated (g) donors.The strength of a correlation (Sperman's correlation coefficient) is demonstrated by the color of the square, and the significance is indicated by asterisks.Correlation between two continuous variables was analyzed using the Spearman correlation analysis.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, no significant difference.
Further support for the protective role of triple-dose CoronaVac comes from our observation that hybrid immunity strengthens and broadens antibody responses, mucosal immunity, and cellular immunity, providing the most sustained protection.Despite there being a large amount of evidence highlighting the remarkable enhance ment of serological and cellular responses elicited by hybrid immunity, there is a paucity of data suggesting that mucosal immunity could also be strengthened.For the first time, our data demonstrate that a larger proportion of breakthrough-infected individuals harbored respiratory spike-specific IgA responses than naturally infected individuals.Additionally, spike-specific nasal IgG and IgA responses of remarkable magnitude were also identified in individuals with hybrid immunity.The augmented mucosal immun ity, especially the nasal IgA response (29), might be strongly associated with superior protection against SARS-CoV-2 reinfection, as described previously (30).
Our study has important implications for the vulnerability of unvaccinated Omicroninfected individuals to reinfection by circulating and emerging SARS-CoV-2.In the absence of vaccination, BA.5 infection-elicited humoral responses showed remarkably reduced serum neutralization activity against BA.5 and BF.7, as well as a subtle decrease against BQ1.1, XBB, XBB.1, and XBB.1.5,and dramatic loss against the ancestral strain.Additionally, despite infection via the respiratory route, the BA.5 spike nasal IgA and IgG titers were dramatically lower in unvaccinated controls than in vaccinated individ uals after BA.5 infection, indicating the possible risk of reinfection.We extended our analysis to Fc-mediated phagocytosis and mucosal immunity.In particular, we found that BA.5-specific ADNP responses and nasal IgA responses were not effectively induced in unvaccinated individuals after BA.5 infection.In contrast, the BA.5-specific T cell response was only subtly lower in unvaccinated individuals than in vaccinated individuals.Taken together, the modest immune responses in BA.5-infected unvaccinated individuals leave this unvaccinated group at risk of being reinfected with Omicron subvariants.Our data indicate that Omicron-based vaccines might not be an ideal immunogen in SARS-CoV-2naive individuals.
For the first time, our results provide an immune landscape of hybrid immunity elicited by BA.5 infection in triple-vaccinated vs unvaccinated individuals.We showed that vaccination with triple-dose CoronaVac effectively evoked higher-quality immune responses characterized by convergent development of cross-reactive humoral and cellular immune compartments, as well as collaborative systemic and mucosal antibody compartments.Machine learning analysis showed that vaccinated individuals generated potent immune responses involving ADCP, ADNP, cTfh cells, activated B cells, and memory B cells, with distinct patterns from unvaccinated donors.Our data also suggest that a one-time Omicron BA.5 infection might not be sufficient to trigger cross-reactive humoral and cellular responses.
This study has some limitations that should be noted.First, T and B cells in the peripheral blood only account for a small proportion of the T and B cell population in the body.Therefore, the measurement of circulating cellular responses might not fully represent the landscape of T cell immunity in vaccinated individuals, especially tissueresident memory T cells.Second, the participants in our cohort were generally young; older individuals were not included.Third, we used peptide stimulation to quantitatively measure T and B cell responses to SARS-CoV-2 spike proteins by flow cytometry.The use of conjugated peptide major histocompatibility complex multimers might result in the sensitive detection of antigen-specific T cells at a higher resolution.Finally, analysis of a more diverse cohort might facilitate further dissection of the immune responses induced by other types of vaccination.
In summary, we have shown that triple-dose CoronaVac remarkably augments antibody, mucosal, and cellular responses that are potent, durable, and cross-reactive to Omicron subvariants.This study sheds light on the dynamics of human adaptive immunity to SARS-CoV-2 Omicron subvariants in a highly vaccinated population with inactivated COVID-19 vaccines.

Study design
The purpose of this prospective observational study (NCT05680896) was to directly compare the humoral and cellular immune responses among triple CoronaVac-vacci nated individuals or unvaccinated individuals who have subsequently contracted the Omicron BA.5 infection during the Omicron wave last December in China.Serum samples were collected from the participants, which were analyzed using ELISA, pseudovirus neutralization assay, antibody-dependent cellular phagocytosis, as well as flow-cytometry-based cellular analysis.Sera were obtained by collecting 4-6 mL of whole blood in a BD Vacutainer Plus Plastic Serum Tube, which was centrifuged for 10 min at 1,000 × g before serum was aliquoted and stored at −20°C.Meanwhile, the nasal swabs were also collected for the measurement of specific mucosal antibody titers.For the cellular analysis, peripheral blood mononuclear cells (PBMC) were isolated from the blood collected in ethylenedia minetetraacetic acid-anticoagulated tubes by lymphocyte separation medium density gradients (Stemcell Technologies, cat# 07801) and resuspended in PRMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 1.5% HEPES buffer (complete medium; Thermo Scientific) for stimulation assays or stored at −135°C until used.

Measurement of serum and nasal SARS-CoV-2 spike-specific humoral responses
Serum or nasal swabs for immunological assessments were taken at three different time points, including 12 months after the third dose before BA.5 infection, 2 weeks, and 20 weeks post-BA.5 infection (4,31).Antigen-specific serological and nasal swab antibodies against SARS-CoV-2 were determined by an in-house enzyme-linked immunosorbent assay, as previously described.Antibody-dependent cellular phagocytosis and antibodydependent neutrophil phagocytosis (ADNP) were described in the previous study (7).The pseudovirus neutralization assay was performed using lentivirus-based SARS-CoV-2 pseudoviruses, which were provided by Vazyme Biotech Co., Ltd, as previously described (4,32).

Antigen-specific measurement of cellular analyses
The biotinylated ectodomain of spike protein was fluorescently labeled to identify SARS-CoV-2-specific circulating B cells and memory B cells.The detailed approach has been described in previous studies (4,5).To measure antigen-specific circulating and memory CD4 + , CD8 + T cells, and cTfh cells, an activation-induced marker assay was performed as described previously (4,5).Stained samples were acquired on a fluorescence-activated cell sorter FACSAria III Cell Sorter instrument (BD Biosciences) and analyzed using FlowJo software version 10.7.1 (FlowJo LLC, BD Bioscience).

Data analysis
To show the potential distinction of immune parameters between two groups, a cold-to-hot hierarchical clustering heatmap represents the scaled magnitude of SARS-CoV-2-specific humoral, mucosal, and cellular immune responses, including spike-binding antibody titers, neutralization activities, Fc-mediated effector function, cellular responses, at T1 time point between vaccinated vs unvaccinated group.Each column represents a vaccinated or unvaccinated individual, while each row represents an immune parameter.The distribution of markers and participants in the cohort was automatically performed by supervised hierarchical clustering.ComplexHeatmap R package (2.15.1) was used for analysis.
A multivariate classification model was built to discriminate immunological profiles among triple vaccinated and unvaccinated groups using tested adaptive immune parameters in our study.All data were normalized using z-scoring before analysis.Feature selection was performed using the least absolute shrinkage and selection operator.Partial least square discriminant analysis (PLS-DA) was used for the classification and visualization of immune parameters from two groups.Selected immune features were ordered based on variable importance in projection score, and the first two latent variables of the PLS-DA model were used for visualization.R package "ropls" version 1.20.043,"glmnet" version 4.0.244, and the systemsseRology R package (v.1.1)(https://github.com/LoosC/systemsseRology)were used for analysis.
For the cross-correlation analysis, Spearman correlation analysis was used for correlation analysis between all tested immune parameters.The correlation analysis was presented using ChiPlot (https://www.chiplot.online/correlation_heatmap.html).

Statistical analysis
Binding antibody titers or neutralizing titers were expressed as geometric mean titers.The mean (standard deviation) or median (95% confidence interval) was used to present the continuous variables.Categorical variables were described as counts and percen tages.Single comparison variables between groups were performed using the Mann-Whitney U test.Multiple comparisons of antibody titers, specific activated or memory B cell and T cell responses were performed using the Friedman's one-way ANOVA with least significant difference (LSD).Correlation between two continuous variables was analyzed using the Spearman correlation analysis.P < 0.05 was considered to be statistically significant.*P< 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, no significant difference.SPSS software program version 22.0 (Chicago, IL, USA) was used for data analysis.

FIG 1
FIG 1 Study design and demographic characteristics of our cohort.(a) Study design of vaccinated individuals and unvaccinated individuals with Omicron BA.5 infection.Vaccination, infection, and blood draw timeline were also noted.(b) The demographic and clinical characteristics of the study cohort.

FIG 3
FIG 3 BA.5 infection enhanced cross-reactive spike-specific B cell responses and circulating follicular helper T (cTfh) cells that persist up to 5 months in vaccinated individuals.(a, c, e, and g) The dynamic frequency of spike-specific B cells (a), memory B cells (c), activated cTfh cells (e), and memory cTfh cells(g) specific to ancestral spike, BA.1 spike, BA.2 spike, and BA.5 spike at T0, T1, and T2 time points.Values above the symbols denote the median, and the percentage of positive responders was also noted.(b, d, f, and h) The comparison of SARS-CoV-2-specific B cells (b), memory B cells (d), activated cTfh cells (f), and memory cTfh cells (h) between vaccinated and unvaccinated individuals at T0, T1, and T2 time points.Fold change of spike-specific B cells between two groups at three time points.Data are shown as the fold change between vaccinated vs unvaccinated donors.Dotted lines indicate the limit of detection for the assay.Single comparison variables between two groups were performed using the Mann-Whitney U test.Multiple comparisons of specific memory B cell responses at three time points were performed using the Friedman's one-way ANOVA with LSD.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, no significant difference.

FIG 4
FIG 4 BA.5 infection enhanced cross-reactive memory T cells among previously triple CoronaVac vaccinees.(a, c, e, and g) The dynamic change of activated CD4+ T cells (a), memory CD4+ T cells (c), activated CD8+ T cells (e), and memory CD8+ T cells (g) specific to ancestral, BA.1, BA.2, and BA.5 spike in vaccinated and unvaccinated groups at T0, T1, and T2 time points.Values above the symbols denote the median, and the percentage of positive responders was also noted.(b, d, f, and h) The comparison of SARS-CoV-2-specific activated CD4+ T cells (b), memory CD4+ T cells (d), activated CD8+ T cells (f), and memory CD8+ T cells (h) between vaccinated and unvaccinated individuals at T0, T1, and T2 time points.Data are shown as the fold change between vaccinated vs unvaccinated donors.Single comparison variables between groups were performed using the Mann-Whitney U test.Multiple comparisons of specific T cell responses of three time points were performed using the Friedman's one-way ANOVA with LSD.Dotted lines indicate the limit of detection for the assay.Bars represent the median value.P < 0.05 was considered to be statistically significant.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, no significant difference.

FIG 5 FIG 6
FIG 5 Increase in the fold of BA.5-specific immune responses post-breakthrough infection was consistently higher than those of ancestral strain.Fold change of serum neutralizing antibody responses (NAb), anti-spike IgG and IgA (a), B cells, memory B cells, cTfh and memory cTfh cells (b), CD4 + T cells, memory CD4 + T cells, CD8 + T cells, and memory CD8 + T cells (c) among vaccinated group against ancestral and BA.5 strain from T1 time point vs T0 time point.Data are shown as the fold change between T0 and T1 in the vaccinated group.Wilcoxon matched-pairs signed rank test was performed for paired data.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, no significant difference.

FIG 6 (
FIG6 (Continued) Healthcare workers at Nanjing Drum Tower Hospital were recruited and enrolled in the study belonging to two groups: vaccinated and unvaccinated individuals.The vaccinated group received the first two doses of CoronaVac in February 2021 and the third dose of CoronaVac in November while the unvaccinated individuals did not receive any COVID-19 vaccine prior to Omicron BA.5 infection.Both groups had PCR-confirmed diagnosis of COVID-19 during the recent Omicron wave from December 2022 and January 2023, and the infected viral sequence was further confirmed by next-generation sequencing.For vaccinated participants, baseline samples were collected at 12 months after the third CoronaVac immunization.The post-infection samples were collected at the median of 18 days (15-21) and 146.5 days (144.8-150.0)post-BA.5 infection in the vaccinated group, and 14 days (13.5-15) and 127.5 days (113.8-140.3) in the unvaccina ted group, respectively.